1. Field of the Invention
The present invention relates to DNA segments which may be employed as functionally translocatable genetic control elements. More particularly, the invention relates to sterol regulatory elements and promoter sequences which serve to promote transcription and/or confer a sterol-mediated suppression capability to selected structural genes.
2. Description of the Related Art
In the 25 years since Jacob and Monod first proposed the lac operon model and the concept of messenger RNA (see, Jacob et al. (1961), J. Mol. Biol., 3: 318-350), the structure and function of a number of prokaryotic operons has been elucidated in elegant detail. For example, in the case of the lac operon, it has been shown that transcriptional control of the various structural genes of the operon (e.g., B-galactosidase) resides in an upstream (i.e., 5' with respect to the structural genes) regulator gene and operator gene. The requlator gene produces a protein "repressor" that interacts with the operator to prevent transcription initiation of the structural gene. Inducers such as IPTG (isopropyl thiogalactoside) bind to the repressor and thereby induce transcription by preventing the binding of the repressor to the operator. Additionally, there is a promoter site P, upstream of the operator and downstream of the regulatory gene, which serves as an RNA polymerase binding site.
Studies on the lac operon further have led to the discovery and elucidation of the mechanism of prokaryotic catabolic suppression. In E. coli it is found that the presence of glucose in the growth medium serves to shut down the expression of gluconeogenic pathways, including the lac operon and its associated structural genes. The mechanism of this catabolic suppression is not entirely clear, but appears to involve a glucose-mediated suppression of cyclic AMP-mediated stimulation of transcription. In this regard, it appears as though cyclic AMP complexes with a protein known as catabolic gene activator protein (CAP), and this complex stimulates transcription initiation. Thus, in the presence of glucose, the activator CAP complex is not formed and transcription is not enhanced.
In addition to the lac operon, the mechanism and structure of numerous additional prokaryotic control mechanisms have been elucidated. (e.g., see Miller et al. (eds.), 1978, The Operon. Cold Spring Harbor Laboratory; Wilcox et al. (1974), J. Biol. Chem., 249: 2946-2952 (arabinose operon); Oxender et al. (1979), Proc. Natl. Acad. Sci., U.S.A., 76: 5524-5528 (trp operon); Ptashne et al. (1976), Science, 194: 156-161 (lambda phage)).
Unfortunately, in contrast to prokaryotic systems, very little is presently known about the control mechanisms in eukaryotic systems. Moreover, although, as noted, the mechanisms for feedback suppression of mRNA production in prokaryotes have been elucidated in elegant detail (see e.g., Ptashne, M. (1986) A Genetic Switch: Gene Control and Phage Lambda. Cell Press and Blackwell Publications, Cambridge, Mass. and Palo Alto, Calif. pp. 1-128), little is known about analogous mechanisms in higher eukaryotes. In animal cells most attention has focused on positively-regulated systems in which hormones, metabolic inducers, and developmental factors increase transcription of genes. These inducing agents are generally thought to activate or form complexes with proteins that stimulate transcription by binding to short sequences of 10 to 20 basepairs (bp) in the 5'-flanking region of the target gene. These elements have been called GRE, MRE, or IRE for glucocorticoid regulatory element, metal regulatory element, and interferon regulatory element, respectively (Yamamoto (1985), Ann. Rev. Genet., 19: 209-252; Stuart et al. (1984), Proc. Natl. Acad. Sci. U.S.A., 81: 7318-7322; Goodbourn et. al. (1986), Cell, 45: 601-610).
Accordingly, there is currently very little knowledge concerning eukaryotic genetic control mechanisms and, in particular, little knowledge concerning negatively controlled genetic elements. The availability of discreet DNA segments which are capable of conferring either a negative or positive control capability to known genes in eukaryotic systems would constitute an extremely useful advance. Not only would such elements be useful in terms of furthering our understanding of eukaryotic gene control in general, but would also provide biomedical science with powerful tools which may be employed by man to provide "fine-tune" control of specific gene expression. The elucidation of such elements would thus provide science with an additional tool for unraveling the mysteries of the eukaryotic gene control and lead to numerous useful applications in the pharmaceutical and biotechnical industries.
Although the potential applications for such control sequences are virtually limitless, one particularly useful application would be as the central component for screening assays to identify new classes of pharmacologically active substances which may be employed to manipulate the transcription of structural genes normally under the control of such control sequences. For example, in the case of hypercholesterolemia, it would be desirable to identify therapeutic agents having the ability to stimulate the cellular production of Low Density LiPoProtein (LDL) receptors, which would in turn serve to lower plasma LDL (and consequently cholesterol) by increasing the cellular uptake of LDL.
Currently, there are few cholesterol-lowering drugs that are both safe and efficacious, and no drugs which are known to operate at the above-described genetic control level. For example, amide from agents that function by sequestering bile salts in the gut and thereby increase cholesterol excretion, the principal therapeutic agent available for cholesterol lowering is dextrothyroxine (Choloxin). Unfortunately, Choloxin causes frequent adverse side effects and, for example, is contra-indicated in ischemic heart disease.
A promising class of drugs currently undergoing clinical investigation for the treatment of hypercholesterolemia acts by inhibiting the activity of HMG CoA reductase, the rate-limiting enzyme of endogenous cholesterol synthesis. Drugs of this class (Compactin and Mevinolin) contain side chains that resemble the native substrate for HMG CoA reductase and that competitively inhibit the activity of the enzyme. Eventually this lowers the endogenous synthesis of cholesterol and, by normal homeostatic mechanisms, plasma cholesterol is taken up by increased LDL receptor populations in order to restore the intracellular cholesterol balance. Conceptually, HMG CoA reductase inhibitors are acting at the penultimate stage of cellular mechanisms for cholesterol metabolism. It would be most desirable if the synthesis of LDL receptor could be directly upregulated at the chromosomal level. The upregulation of LDL receptor synthesis at the chromosomal level offers the promise of resetting the level of blood cholesterol at a lower and clinically more desirable level (Brown et al. (1984), Scientific American, 251:58-60). However, no methods exist for conveniently assaying the ability of a candidate composition to exert such an effect on the transcription of LDL receptor DNA.
Accordingly it is a further object herein to provide a method for conveniently evaluating candidate substances for receptor upregulating activity.